Application of tryptic condensation strategy for the synthesis of α-melanocyte stimulating hormone

The use of trypsin in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) paired with N,N-dimethylformamide (DMF) has been proposed for the enzymatic condensation of peptides. The tryptic condensation strategy was applied to the synthesis of the 13-peptide, α-melanocyte stimulating hormone (α-MSH), which contains two susceptible points to trypsin, in a low-water-containing solvent system, 4% H₂O in HFIP/DMF (1/1, v/v). The N-terminal 8-peptide segment (S1) ending with Arg and C-terminal 5-peptide with the side-chain protections at Lys and Trp (S2) were prepared by the stepwise coupling on p-nitrobenzophenone oxime resin and by a conventional method, respectively. Both segments were condensed by the aid of trypsin. The acid component was converted into the partially protected α-MSH at as high as 95% conversion determined by reversed-phase HPLC. When the side-chain of Lys at the 11-position was not protected, α-MSH was obtained only in 35% yield, and was contaminated with products of secondary hydrolysis. Although the Lys¹¹-Pro¹² sequence was very poorly susceptible to trypsin, the side-chain of Lys had to be protected in order to be inert to trypsin under the synthetic conditions. HFIP is demonstrated as a good solvent with DMF to allow the efficient tryptic condensation of peptides. The strategy increased the value of the enzymatic condensation as practical method by avoidance of secondary hydrolysis, high dissolution of peptides and retention of activity of enzyme.     

Synthesis of phospho-urodilatin by combination of global phosphorylation with the segment coupling approach

The chemical synthesis of biologically active phosphorylated urodilatin (CDD/ANP-95-126) was achieved by using a strategy of coupling protected peptide segments in solution. Three protected peptide segments corresponding to urodilatin (1-14) with side chain-unprotected Ser¹⁰, (15-24) and (25-32) were prepared manually using Fmoc chemistry on an aminopropyl polystyrene resin with the super acid-labile HMPB linker. For the coupling of segments, the carboxy group of the C-terminal segment (25-32) was converted into the tert-butyl ester by treatment with TBTA. The protected peptide segments were coupled in the presence of EDC/HOOBt or TBTU/HOBt to yield fully protected urodilatin with a free hydroxy function at Ser¹⁰. Introduction of the phosphate was performed with Et₂NP(OtBu)₂ and tetrazole followed by oxidation of the phosphite. Alternatively, a prephosphorylated protected segment (1-14) was used in the segment condensation. Our investigations indicate that both pathways, phosphorylation of protected urodilatin in solution and use of a prephosphorylated building block, are suitable methods to obtain a large phosphopeptide of high purity without formation of H-phosphonates or other by-products. © Munksgaard 1996.     

Solution structure of synthetic peptide inhibitor and substrate of cAMP-dependent protein kinase. A study by 2D 1H NMR and molecular dynamics

Peptides derived from the inhibitor of cAMP-dependent protein kinase, PKI, have been studied by 2D ¹H NMR techniques. These include the inhibitor PKI(6-22), the substrate [Ala²⁰-Ser²¹] PKI(5-24), and a phosphorylated form of the latter [Ala²⁰-Ser²¹P] PKI(5-24). A homologous fold was found in the three peptides which consisted of an N-terminal segment in helical conformation to residue 13 and a C-terminal segment poorly defined conformationally. A parallel study was carried out by molecular dynamics (MD) for the inhibitor peptide PKI (5-24). The N-terminal helix, as observed in the crystal structure of the catalytic subunit-PKI(5-24) complex, was conserved in the MD simulations with the enzyme-free inhibitor. Similarly the Gly¹⁴-Gly¹⁷ turn was apparent in all MD structures, whereas the C-terminal region, residues 18-24, was directed towards the N-terminal helix in contrast to the extended conformation of this segment pointing away from the N-terminal helix in the crystal structure. This is primarily due to ionic interaction between Asp⁹ and Arg¹⁵. Indeed, a detailed analysis of the NOE contacts by NOESY at low temperature (2°C) shows the occurrence of pH-dependent contacts with Phe¹⁰. We conclude that the binding of short inhibitors, such as PKI (5-24), to the enzyme involves a conformational rearrangement of the C-terminal region. The substrate [Ala²⁰-Ser²¹] PKI (5-24) and the product [Ala²⁰-Ser²¹P] PKI(5-24), give very similar structures with local rearrangements involving some of the side chains. © Munksgaard 1997.     

Racemization free coupling of peptide segments

The total synthesis of the insect neuropeptide derivative Z-Gly-Gly-Ser-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂ has been carried out by a convergent solid phase strategy. For the coupling of the N-terminal pentapeptide to the C-terminal tetrapeptide, three different methods were assayed. Racemization of the acyl activated amino acid during the fragment condensation reaction was monitored by HPLC. Best results were obtained by enzymatic coupling in a low water containing media using adsorbed α-chymotrypsin. An optically pure product was obtained in 82% yield after 1 h of reaction. Chemical methods such as DIC/HOBt and BOP/HOBt NMM always rendered highly optically impure products containing 10-20% of the d -epimer.     

Solution synthesis of the glyco-hex a peptide sequence of the human oncofetal fibronectin defined by monoclonal antibody FDC-6

The glyco-hexapeptide sequence H-Val-(GalNAc-α)Thr-His-Pro-Gly-Tyr-OH, was synthesized in solution by the segment condensation procedure and the stepwise procedure. A peracetylated, O-galactosaminyl threonine derivative was used for incorporating the glycosylated amino acid residue into the peptide chain. A consistent racemization occurred during the acylation of H-His-Pro-Gly-Tyr(Bzl)-OBzl with Z-Val-[GalNAc(Ac)₃-α]Thr-OH by the BOP-HOBt procedure and the D-allothreonine containing glycohexapeptide was isolated in about 20% yield. Stepwise elongation of the C-terminal tetrapeptide with Frnoc-[GalNAc(Ac)₃-a]Thr-OH and Z-Val-OH, in the presence of the same coupling reagents, yielded the l -threonine containing diastereoisomer without detectable racemization. A side product, the N^im-ethoxy-carbonylated hexapeptide derivative, formed during the EEDQ-mediated condensation of Fmoc-[GalNAc(Ac)₃-α]Thr-OH with the C-terminal tetrapeptide, was isolated and characterized. Preliminary studies showed that the synthetic glycohexapeptide is a good competitive inhibitor of the binding of the FDC-6 monoclonal antibody to the oncofetal fibronectin, supporting the idea that it should represent the minimum essential structure required for the FDC-6 activity.     

Synthesis of human angiotensinogen (1-17) containing one of the putative glycosylation binding sites and its hydrolysis by human renin and porcine pepsin

The N-terminal heptadecapeptide of human angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-Glu-Ser-Thr-NH₂), with the C-terminal carboxyl group amidated, was synthesized in order to study the role of Asn-Glu-Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h-angiotensinogen (1-17)-NH₂ with a K_m value of 3.4 × 10^?5m , at pH 7.3 and 37° being similar to h-angiotensinogen (1-13), an analog without the carbohydrate binding site. However, the V_max value of 4.1 × 10^?9mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu¹⁰-Val¹¹ bond and, surprisingly, His⁹-Leu¹⁰ as well.     

Resolution of proline acylation problem for thiol capture strategy by use of a chloro-dibenzofuran template

The acyl transfer rate for proline, in the prior thiol capture strategy, was enhanced by changing the electronic character of the dibenzofuran template. The rate of amide bond formation between proline and cysteine by the 1-chloro-4-hydroxy-6-mercaptodibenzofuran was measured to be 0.012 min^-1, which translates to a half-life of 53 min. Further enhancement of the reaction rate was accomplished by the use of a 1,3-dichloro-dibenzofuran template. The k₁ for the reaction was measured to be 0.093 min^-1, and the half-life was calculated to be 7 min. To test the applicability of the activated template, 1-chloro-4-hydroxy-6-mercaptodibenzofuran, in peptide synthesis, the 34 amino acid long peptide, H-RPDFCLEPPYTGPCRKARNNFKSADECMRTCGGA-OH, was synthesized. This peptide represents the condensation of the N-terminal 13-mer and the C-terminal 21-mer of the basic pancreatic trypsin inhibitor.     

Self-association and solubility of peptides.

Self-associated species of the protected C-terminal tetrapeptide segment of porcine vasoactive intestinal peptide and related short sequences in methylene chloride have been disrupted by adding increasing amounts of dimethylsulphoxide. This structural transition has been monitored following the disappearance of the amide I carbonyl stretching band assigned to strongly intermolecularly hydrogen-bonded molecules (1655–1633 cm^-1) in the infrared absorption spectra. A scale for the propensity of the various peptides to aggregate has been established. Substitution of the asparagine residue by a β-tert.-butylaspartic acid residue in the tripeptide drastically reduces the extent of self-association. The increasing tendency to aggregate shown by these peptides is paralleled by a decrease in their solubility. The impact of these results on the strategy of synthesis of porcine vasoactive intestinal peptide is briefly outlined.     

Design,synthesis and antibacterial activity of cecropin-like model peptides

In order to investigate structure-activity relationships of cecropins, model peptides that mimic certain structural features of the cecropin molecules were designed and synthesized. The conformational analysis of cecropins and the design of the model peptides were based on Chou-Fasman calculations. The peptides were synthesized by solid-phase methods and purified by reverse-phase liquid-chromatography on C₁₈-silica columns. Their secondary structures were studied by circular dichroism measurements. Antibacterial activities against seven test organisms were determined and compared to the activities of the natural cecropins A and B. These results were discussed on the basis of structural features of the model peptides and on model mechanisms. It was concluded that high antibacterial activity for this class of compounds requires a basic helical amphipathic N-terminal segment that is connected to a hydrophobic helical C-terminal segment by a flexible non-helical hinge region.     

Synthesis,conformation, and biological activity of the carbohydrate-free vespulakinin 1

Synthesis of the carbohydrate-free heptadecapeptide corresponding to the amino acid sequence of vespulakinin 1 was achieved by the continuous flow solid phase procedure on 4-hydroxymethyl-phenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin, as well as by a combination of solid phase and solution syntheses. Preformed Fmoc-amino acid symmetrical anhydrides (Boc derivative for the N-terminal residue) were used for amine acylation in the continuous flow method. Serine and threonine were side chain protected as tert.-butyl ethers and the 4-methoxy-2, 3, 6,-trimethyl-benzenesulfonyl group was used for masking the guanidino function of arginine residues. After cleavage from the resin the final peptide was purified by ion exchange chromatography and characterized by amino acid analysis, high voltage electrophoresis, and RP-HPLC analysis. Alternatively, the protected N-terminal octapeptide, Fmoc-Thr(Bu^t)-Ala-Thr(Bu^t)-Thr(Bu^t)-Arg(Mtr)-Arg-(Mtr)-Arg(Mtr)-Gly-OH was prepared on 4-hydroxymethyl-3-methoxyphenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin and the C-terminal nonapeptide H-Arg(NO₂)-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-(NO₂)-OBzl was synthesized in solution through the fragment condensation method. The two fragments were coupled by the DCC-HOBt procedure and the resulting heptadecapeptide was deblocked and purified. The conformational features of the synthesized peptides are reported. Preliminary pharmacological experiments indicated that carbohydrate-free vespulakinin 1 is more potent than bradykinin in lowering rat blood pressure.     

Binding of C-terminal segments of neuropeptide Y to chicken brain

The C-terminal pentapeptide amide segment of neuropeptide Y (NPY) binds specifically to chicken brain membrane preparations. The contribution of each residue of the C-terminus to this binding has been investigated through the synthesis and evaluation of a series of pentapeptide analogs. The binding of these molecules is strongly dependent on the presence of certain functional groups, in particular the guanidinium group of Arg-35 and the C-terminal aromatic amide function. The significance of these results for the binding to chicken brain tissue of NPY, pancreatic polypeptide and other potentially related neuropeptides is discussed.     

Orthogonal solid-phase synthesis of human gastrin-I under mild conditions*

An approach to the solid-phase segment condensation synthesis of the 17-peptide amide human gastrin-I has been developed. N^α-amino and side-chain protection were provided by 9-fluorenylmethyloxycarbonyl (Fmoc) and tert.-butyl groups, and a series of anchors cleavable under mild conditions were used. The N-terminal pentapeptide pGlu-Gly-Pro-Trp-Leu-OH was prepared using a p-alkoxybenzyl ester linkage made by a preformed handle strategy. Cleavage, in 65% yield, was with the new Reagent M: CF₃ COOH—CH₂ Cl₂—β-mercaptoethanol-anisole (70:30:2:1), which was optimized to preserve the labile tryptophan residue. A new preformed handle procedure expedited solid-phase synthesis of the protected “middle” hexapeptide, Fmoc-(Glu(OtBu))₅-Ala-OH, anchored as an o-nitrobenzyl ester. Chains were not lost during this assembly, and final photolytic cleavage (350nm) in toluene—CF₃ CH₂ OH (4:1) occurred in 59% yield. Both protected intermediates were purified by simple gel filtration, whereupon they were shown to be pure by analytical HPLC, and gave satisfactory NMR and FABMS spectra. Last, the C-terminal hexapeptide, Tyr(tBu)-Gly-Trp-Met-Asp(OtBu)-Phe, was assembled on a tris(alkoxy)benzylamide “PAL” support. For the polymer-supported segment condensation, the middle and N-terminal pieces were added respectively in > 98% and 89% yields (judged by amino acid analysis and solid-phase sequencing), by overnight couplings in N,N-dimethylformamide (DMF) mediated by benzotriazolyl N-oxytrisdimethylaminophosphonium hexafluorophosphate (BOP) in the presence of 1-hydroxybenzotriazole (HOBt) and N-methylmorpholine (NMM). Racemization was 4% and 11% respectively at Ala and Leu. Cleavage with Reagent M followed by reversed-phase chromatography gave pure gastrin-I in an overall 30% isolated yield. These results compare favorably with those from a stepwise assembly.     

Solid-phase synthesis of peptides with C-terminal asparagine or glutamine

Attempts to anchor Fmoc-asparagine or glutamine as p-alkoxybenzyl esters for solid-phase peptide synthesis are fraught with difficulties. A convenient and effective method to prepare peptides with C-terminal asparagine or glutamine involves quantitative attachment of N^x -Fmoc-C^x -tert.-butyl aspartate or glutamate via the free ω-carboxyl groups to a tris(alkoxy)benzylamino (PAL) support. Chain elongation proceeds normally by standard Fmoc chemistry, and treatment with acid, e.g., CF₃COOH—CH₂Cl₂, 90 min at 25°, releases the desired peptides in >95% yields without side reactions at the C-terminus. Feasibility of the approach has been demonstrated by the syntheses of the C-terminal octapeptide from human proinsulin, H-Leu-Ala-Leu-Glu-Gly-Ser-Leu-Gin-OH, and the serum thymic factor pGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH.     

Photochemical conjugation of peptides to carrier proteins using 1,2,3-thiadiazole-4-carboxylic acid Immunoreactivity of free C-terminal epitope with specific antibodies

A new heterobifunctional cross-linking reagent, 1,2,3-thiadiazole-4-carboxylic acid, for the photochemical conjugation of peptides to proteins is described. The title compound can be coupled directly to a protected peptide resin during solid-phase peptide synthesis (SPPS) using standard coupling procedures. The probe is stable to TFA deprotection/cleavage mixtures containing ethanedithiol commonly used in Fmoc-SPPS. Furthermore, tritium may easily be introduced into the thiadiazole ring by base-catalyzed hydrogen-exchange. Upon irradiation at 245-300 nm, parent 1,2,3-thiadiazole rapidly eliminates N₂, generating very reactive thioketene which reacts with amines to give a thioamide in high yield, even when the photolysis is carried out in hydroxylic solvents. In order to investigate the potential of the title compound as a heterobifunctional cross-linking reagent a model study with angiotensin II (AII) was conducted. The photoreactive peptide N^α-4-carbonyl-1,2,3-thiadiazole-AII (TDA-AII) was synthesized by Fmoc-SPPS and conjugated to bovine serum albumin (BSA) by photolysis at 245 and 300 nm. By use of a capture competition ELISA, the C-terminal Pro-Phe epitope of photoconjugated AII with the sequence DRVYIHPF was shown to bind specifically to antiAII antibodies (anti-AII abs), although antibodies against both the C- and N-terminal epitopes were present in the assay. A dipeptide His-Leu carboxy-extension form of AII, angiotensin I (AI), only bound to anti-AII abs at 100-200 times higher concentrations, showing that the C-terminal epitope was blocked by the dipeptide. © Munksgaard 1996.     

Solution structure of the phosphorylated sites of ribosomal protein S6 by 1H NMR spectroscopy

An increase in the rate of protein synthesis is found to be accompanied by phosphorylation of the 40S ribosomal protein S6. Treatment of S6 by cyanogen bromide produced three fragments, and one of the fragments of S6, which is a C-terminal portion of S6 (M_r? 4000), contains all phosphorylation sites of S6. The C-terminal fragment of S6 contains seven serines. S6 kinase phosphorylates S6 specifically, i.e. five serines in the C-terminal of S6 are phosphorylated. The three-dimensional structure of S6 peptide was studied in 50% trifluoroethanol/50% H₂O solution by ¹H NMR with combined use of distance geometry and restrained molecular dynamics calculations. NMR results indicated that it takes an α-helix between Glu5 and Arg21 and a distorted helical structure for the following three residues, but no rigid structure was present from Ser25 through the C-terminus and for the N-terminal region (Lys1-Lys4). The specificity of the phosphorylation of the peptide is discussed from a structural aspect. © Munksgaard 1996.     

Isolation and identification of C-terminal peptides of proteins

A method for the isolation and identification of C-terminal peptides of proteins has been developed. The procedure entails the racemization of the C-terminal amino acid by reaction of the N-trifluoroacetylated protein with acetic anhydride and pyridine. After deprotection, the protein is fragmented to yield a mixture of peptides, which in turn are digested with carboxypeptidases. All peptides are hydrolyzed to L-amino acids except the C-terminal peptide with the terminal D-amino-acid residue. It is resistant to the action of carboxypeptidases and is readily identified by peptide mapping. © Munksgaard 1995.     

Small-scale manual multiple peptide synthesis system

A system for small-scale (ca. 10–50 μmol) manual multiple peptide synthesis assembled from commercially available solid-phase extraction apparatus is described. This system was used to prepare (on a 15 μmol scale) the five monophosphorylated isomers of the peptide ASTTVSKTE, a proteolytic fragment of the C-terminal region of rhodopsin. The peptides were assembled using serine or threonine active esters without hydroxyl protection at the positions of phosphorylation. Phosphate groups were introduced using postassembly phosphitylation/oxidation according to a published procedure [Andrews, D.M., Kitchin, J. & Seale, P.W. (1991) Int. J. Peptide Protein Res. 38, 469–475; Staerkaer, G., Jakobsen, M.H., Olsen, C.E. & Holm, A., Tetrahedron Lett. 32, 5389–5392; Perich, J.W. (1992) Int. J. Peptide Protein Res. 40 134–140], The reported system provides a relatively inexpensive approach to multiple peptide synthesis, including synthesis of phosphopeptides, for laboratories whose synthesis requirements do not justify investment in an automated multiple peptide synthesis instrument.     

Amidation of growth hormone releasing factor (1–29) by serine carboxypeptidase catalysed transpeptidation

The applicability of serine carboxypeptidase catalysed transpeptidation reactions, using amino acid amides as nucleophiles, for C-terminal amidation of peptides has been investigated. With the aim of converting an unamidated precursor into GRF(1–29)-NH₂, an interesting biologically active derivative of growth hormone releasing factor, a number of model reactions were initially investigated. In such a transpeptidation reaction, where the C-terminal amino acid is replaced by the amino acid amide, used as nucleophile, the C-terminal amino acid residue of the substrate can be chosen freely since it functions as leaving group and does not constitute part of the product. Since the C-terminal sequence of GRF(1–29)-NH₂ is -Met-Ser-Arg-NH₂ the model reactions Bz-Met-Ser-X-OH (X = Ala, Leu, Arg) + H-Arg-NH₂→ Bz-Met-Ser-Arg-NH₂+ H-X-OH were first studied. With carboxypeptidase Y and X = Ala or Leu the amidated product could be obtained of 98% and 41%, respectively. With carboxypeptidase W-II and X = Arg a yield of no more than 72 % could be obtained. The choice of Ala as leaving group in combination with carboxypeptidase Y therefore appeared optimal. With the longer peptide Bz-Leu-Gln-Asp-Ile-Met-Ser-Ala-OH the amidated product could be obtained in a yield of 78%, using carboxypeptidase Y, the only other product being Bz-Leu-Gln-Asp-Ile-Met-Ser-OH, formed due to the competing hydrolysis reaction. The full length peptide GRF(1–28)-Ala-OH was synthesized by the continuous flow polyamide solid-phase method. With this peptide the yield of amidation was 87 % and the remaining products were GRF (1–28)-OH and GRF(1–28)-Arg-Arg-NH₂. However, these can be separated from the desired product by ion exchange chromatography. The results demonstrate that it is feasible to use the generally stable and easily accessible serine carboxy-peptidase for C-terminal amidation of peptides, e.g. produced by fermentation of genetically manipulated microorganisms.     

Structure-activity studies on C-terminal hirudin peptides containing sulfated tyrosine residues

To clarify the role of the negative charge of the C-terminal region of hirudin, we chemically synthesized the C-terminal peptide of hirudin variant-1 (HV-1), HV-1-(54-65), and its analogs, [E61Y,E62Y]HV-1-(54-65) and [E62Y]HV-1-(54-65), and then sulfated the Tyr residue(s) in these peptides by both enzymic and chemical methods. Enzymic O-sulfation of Tyr residues in the peptides by use of sulfotransferase isolated from Eubacterium A-44 allowed us to produce four kinds of the sulfated peptide, whose C-terminal sequences were -PEY(SO₃H)YLQ, -PYY(SO₃H)YLQ, -PYYY(SO₃H)LQ and -PYY(SO₃H)Y(SO₃H)LQ. On the other hand, all Tyr residues in the peptides were successfully sulfated by chemical reaction with N, N′-dicyclohexylcarbodiimide in the presence of sulfuric acid. Based on the analysis of structure-activity relationships of these sulfated peptides for thrombin inhibition, the Tyr62 and Tyr63 bisulfated peptide GDFEEIPEY(SO₃H)Y(SO₃H)LQ was found to be the most potent inhibitor of thrombin among the products tested. No increase in potency was observed by further substitution of Glu61 with Tyr(SO₃H). The inhibitory activity by substitution with Tyr(SO₃H) at position 63 was greater than that obtained by the substitution at position 62. © Munksgaard 1996.     

Synthesis of histidine-containing dipeptide affinity-labelling agents

Peptidyl diazomethyl ketones and fluoromethyl ketones containing histidine in the C-terminal position were synthesized to determine their properties as proteinase inactivators. These were examined chiefly with derivatives of Z-Ala-His. The protection of histidine during conversion of the C-terminal residue to the diazomethyl ketone required unblocking conditions which avoid acid due to the lability of this function. This was achievable with a Cbz-imidazole derivative since aminolysis provided deblocking without disturbance of the diazomethyl ketone function. In the case of the fluoromethyl ketone synthesis using fluoroacetic anhydride (Dakin-West procedure), the desired product could be isolated without ring blocking. The Z-Ala-His products showed enhanced selectivity for inactivation of cathepsin B over L when compared to analogous dipeptide inhibitors.